This invention relates to the field of nucleic acid detection and, more specifically, to the preparation of stabilized cocktails of reagents for nucleic acid amplification.
Nucleic acid detection through modern molecular biological techniques has revolutionized diagnosis of infections, cancer, inborn genetic errors, HLA typing, and forensic and paternity testing. Diagnosis is accomplished through any of a variety of nucleic acid detecting methods, including, for example, the polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription mediated amplification (TMA) reaction, nucleic acid sequence based amplification (NASBA) reaction, and strand displacement amplification (SDA) reaction.
Reagents used in nucleic acid detection methods are typically prepared separately as individual stock solutions and are combined to produce the cocktail just prior to its use. For example, in PCR, a cocktail of reagents contains a DNA polymerase, appropriate nucleoside triphosphates, primer(s), and an amplification buffer. Typically, the cocktail of reagents cannot be stored at 4xc2x0 C. for an extended period of time, but must be made fresh just before use to avoid undesirable reactions during storage between the individual reagents such as non-specific DNA polymerization of the nucleoside triphosphates in the absence of a target template.
The requirement to prepare and quality control separate stock solutions of each reagent used in amplification increases the costs of nucleic acid detection in the clinical lab. Also, the requirement to add several reagents to make the cocktail just before use increases the likelihood of error in the clinical lab. Thus, there is a need for a stabilized cocktail of nucleic acid detection reagents that is stable for extended times at 4xc2x0 C.
Accordingly, it is an object of the present invention to eliminate the requirement for separate preparation and quality control of each reagent used in a nucleic acid amplification reaction by providing a cocktail of the reagents in which undesirable reactions during storage between the reagents are avoided.
To accomplish this and other objectives, there has been provided, according to one aspect of the present invention, a composition comprising a cocktail of reagents for performing nucleic acid amplification that avoids undesirable reactions during storage between the individual reagents, thereby stabilizing the cocktail upon storage, comprising one or more of the reagents necessary to perform nucleic acid amplification and an inhibitory concentration of a reversible inhibitor(s) of the undesirable reaction.
According to one embodiment, the cocktail of reagents comprises one or more of a nucleic acid polymerase or ligase and one or more of a nucleoside triphosphate(s), nucleic acid primer(s) and an amplification buffer.
According to another embodiment, the cocktail of reagents comprises a lipid, which can be in the form liposomal vesicles wherein the cocktail of reagents is encapsulated within the liposomes.
According to yet another embodiment, the cocktail of reagents comprises all the reagents necessary to perform a nucleic acid amplification reaction.
According to another embodiment, the inhibitor of the undesirable reactions upon storage is a nucleic acid binding ligand. The binding ligand can be an intercalator compound, which can be monoadduct forming. The intercalator compound can be a furocoumarin such as 4xe2x80x2-aminomethyltrioxsalen (xe2x80x9cAMTxe2x80x9d) or angelicin, or a phenanthridine. The binding ligand also can be a non-intercalating compound such as benzimides, netropsins and distamycins.
According to another embodiment of the present invention, a method of nucleic acid amplification is provided using the composition comprising a stabilized cocktail of reagents.
According to yet another embodiment of the present invention, a method for preparing a stabilized cocktail of reagents which avoids undesirable reactions that occur between the reagents upon storage is provided. The method includes adding the inhibitor(s) of the undesirable reactions to the cocktail of reagents, wherein the inhibitor is added to the cocktail at a concentration that is inhibitory to the reaction but at a concentration which will be non-inhibitory when the cocktail is later diluted for its intended use. The method further includes adding a lipid for releasing nucleic acid from cells. In such cases, the lipid is used to produce liposomal vesicles and the stabilized cocktail of reagents and the inhibitor are encapsulated within the vesicles.
According to still yet another embodiment, the method for preparing a stabilized cocktail of reagents includes reagents suitable for performing polymerase chain reaction, ligase chain reaction, transcription based amplification reaction, nucleic acid sequence based amplification reaction and strand displacement amplification reaction.
According to another embodiment of the invention, the method of preparing a stabilized cocktail is for a transcription based or amplification reaction or a ligase chain reaction and said inhibitor(s) is phosphate ion.
According to yet another embodiment, the method for preparing a stabilized cocktail includes a binding ligand as the inhibitor. The binding ligand can be an intercalator compound, which can be monoadduct forming. The intercalator compound can be a furocoumarin such as AMT. The binding ligand also can be a non-intercalating compound.
According to still yet another aspect of the present invention, kits for performing nucleic acid amplification using the stabilized cocktail of reagents are provided.
The present invention provides novel compositions and methods for preparing a cocktail of reagents that avoids undesirable reactions during storage between the reagents by addition of a reversible inhibitor of the reaction. Such undesirable reactions include, for example, formation of primer dimers, degradation of primers by exonuclease activity of the polymerase and non-specific polymerization of nucleoside triphosphates and/or primers.
The reagent cocktail is stable because of the presence of the inhibitor, thus allowing the cocktail to be stored for later use in amplification. Amplification is achieved when the cocktail is appropriately diluted with the target template such that the concentration of reaction inhibitor is below its effective level while the concentration of the other reagents are at an effective level. The use of stabilized cocktail of reagents eliminates the cost of preparation and quality control associated with preparing individual stock solutions of each reagent required for a particular nucleic acid extraction and/or detection.
General Definitions
Oligonucleotide: Low molecular weight deoxyribo-, ribo-, copolymers of deoxyribo- and ribonucleic acids of chain lengths between 3 and 150. Such oligonucleotides can have modified nucleotide residues such as xe2x80x94O-methoxy, phosphorothio-, methylphosphonates and others known in art.
Primers: Usually oligonucleotides which are used for extension reaction by a nucleic acid polymerase after a template primer hybrid is formed. Such primers can carry sequences specific for transcription by an RNA polymerase.
Nucleic Acid Probe: Nucleic acid with substantially complementary sequences to the target nucleic acids for detection or capture from a mixture. Such probes can be labeled for detection or immobilized onto a solid support to enrich the target by capture. A probe can be an single stranded or partially double stranded and can be an oligonucleotide or a larger nucleic acid.
Membrane fluidizing compound: A chemical substance that renders a cell membrane fluid or flexible to facilitate release of cellular material into solution or uptake of extracellular contents. Compounds that induce pinocytosis in addition to fluidizing the membrane also are included within the meaning of a membrane fluidizing compound as used herein. A membrane fluidizing compound can be a lipid or a non-lipid and can be ionic or non-ionic. Membrane fluidizing compounds generally do not cause cell death at lower concentrations that effect membrane fluidity, however, cell death typically results at higher concentrations of the compound.
Lipid: Any of various substances that are soluble in non-polar organic solvents (such as chloroform and ether), that with proteins and carbohydrates constitute the principal structural components of living cells, and that include fats, waxes, phosphatides, cerebrosides, and related and derived compounds.
Liposome vesicles: A vesicle composed of one or more concentric phospholipid bilayers. The structure of the liposomes may be as a multilamellar vesicle (MLV), a small unilamellar vesicle (SUV), a large unilamellar vesicle (LUV). A liposome is formed from a single lipid or combination of lipids (i.e., lipsosmal formulation) and optionally other compounds.
Thiocationic lipid: A lipid molecule with sulfur substitution and which is positively charged at neutral pH.
Photoreagent or photoactive reagents: Reagents which under appropriate wavelengths of light exposure form a covalent bond with nucleic acid.
Preferred Embodiments
The present invention provides compositions comprising a cocktail of reagents for performing nucleic acid amplification that avoids undesirable reactions during storage between the individual reagents, thereby stabilizing the cocktail upon storage. Such composition comprises one or more of the reagents necessary to perform nucleic acid amplification and an inhibitory concentration of a reversible inhibitor(s) of the undesirable reaction. The inhibitor is added to the cocktail at a concentration that is inhibitory to the reaction, but at a concentration that will be non-inhibitory when the cocktail is later diluted for its intended use. The cocktail of reagents generally includes a nucleic acid polymerase, a reversible inhibitor(s) of the undesirable reaction and one or more of a nucleoside triphosphate(s), nucleic acid primer(s) and an amplification buffer.
Inhibitors of amplification reactions which are suitable for use in stabilizing a cocktail of amplification reagents include, for example, reagents well known in the art as amplification inhibitors. For example, phosphate ion is inhibitory for a transcription mediated amplification reaction (Della-Latta, et al., J. Clin. Microbiol., 37:1234-1235 (1999)). An inhibitory concentration of phosphate ion for a transcription mediated reaction is about 0.7 mM. In addition, phosphate ion above 1.2 mM is inhibitory for a ligase chain reaction (Notomi, et al., J. Clin. Pathol., 51:306-308, (1998)). Certain polysaccharides, heme and components present in urine also inhibit amplification reactions (Mahony, et al., J. Clin. Microbiol., 36:3122-26 (1998); Moreira, Nucleic Acids Res., 26(13):3309-10 (1998)). These reversible inhibitors can be added to cocktails in the present invention to stabilize the components upon storage prior to use of the cocktail in amplification as described above.
Stabilized amplification cocktails of the invention are stable upon storage at 4xc2x0 C. for 24 hours (hrs), more preferably for 48 hrs, still more preferably for 72 hrs and most preferably for more than one week. Stabilized amplification cocktails of the invention also are stable upon storage at 25xc2x0 C. preferably for 8 hrs and more preferably for 24 hrs.
The present invention provides a composition comprising a cocktail for amplification containing 250 mM of phosphate ion. In this mixture no amplification will occur and all the reagents will remain inactive and stable. Amplification can later be achieved by diluting the composition ten fold during preparation of an amplification reaction mixture. At 25 mM phosphate, the amplification reaction will not be inhibited.
The present invention also provides compositions for stabilizing a cocktail where the reversible inhibitor has nucleic acid binding properties such as intercalators like furocoumarins, phenanthridines, acridines, phenazines or non-intercators like netropsin, distamycin and others. Representative intercalating agents suitable as inhibitors include azidoacridine, ethidium monoazide, ethidium diazide, ethidium dimer azide (Mitchell, et al., J. Am. Chem. Soc., 104:4265 (1982)), and 4-azido-7-chloroquinoline, and 2-azidofluorene. A specific nucleic acid binding azido compound has been described by Forster, et al., Nucleic Acid Res., 13:745 (1985). Such compounds include nucleic acid binding ligands as described herein for labeling nucleic acid (i.e., light activated compounds: xe2x80x9cLACsxe2x80x9d). The present invention provides a method for reversibly inhibiting a PCR amplification using the inhibitor, 4xe2x80x2-aminomethyltrioxsalen (xe2x80x9cAMTxe2x80x9d), which is a nucleic acid binding ligand.
Inhibitors that are DNA binding ligands also can include additional substituents that are useful for other aspects of nucleic acid detection, provided that the substituents do not impair the inhibitory nature of the compound. For example, photoreactive forms of intercalating agents such as the azidointercalators are useful as both an inhibitor of the undesirable reaction in reagent preparation and for labeling a nucleic acid covalently upon photoactivation. Other useful inhibitors that are photoreactable intercalators include the furocoumarins which form (2+2) cycloadducts with pyrimidine residues, alkylating agents such as bis-chloroethylamines and epoxides or aziridines, e.g., aflatoxins, polycyclic hydrocarbon epoxides, mitomycin and norphillin A.
Specific LACs which can be used as reversible inhibitors to stabilize amplification cocktails include, 4xe2x80x2-Biotinyl-PEG-4,5xe2x80x2-dimethylangelicin (BPA: Example 17)), Angelicin-DAPI-Biotin (BDA: Example 21)), bisbenzimidazole-PEG-azidonitrobenzene (AZPIMA: Example 20)), Angelicin-bisbenzimidazole-PEG-acridine (xe2x80x9cAPIMAxe2x80x9d), Angelicin-bisbenzimidazole-PEG-biotin (BPIMA: Example 19) and compounds described in U.S. Pat. Nos. 4,950,744 and 5,026,840. In such compounds, PEG represents any molecular weight or polymer substituent that is known to comprise polyethyleneglycol, including pentaoxaheptadecane.
The desired concentration of each inhibitor in the composition for stabilization can be determined by one skilled in the art using known methods. For example, it is helpful to first determine the concentration of inhibitor at which inhibition of undesirable reactions during storage occur and the concentration at which the inhibitor does not affect labeling. Once the dilution range between inhibition and non-inhibition is established, this dilution then dictates the fold concentration necessary of the cocktail of labeling reagents. The concentration of the labeling reagent in the mixture is preferably about 10 fold higher than the concentration at which the inhibitor allows an amplification reaction to yield a detectable amplification product, although concentrations of 20 fold to 50 fold also are useful.
In some embodiments, the cocktail of reagents also includes a lipid to form a liposomal vesicle or other structure to encapsulate the cocktail. In this approach, the microenvironment of the liposomal vesicle or other structure allows the inhibitor to be at a sufficiently high concentration to stabilize the reagents. When the cocktail including the lipid is used for amplification, it is diluted such that the vesicles or other structure is disrupted, thus releasing the reagents and reducing the inhibitor concentration below that which causes inhibition.
Prior methods of forming liposomes and encapsulating aqueous solution are applicable for preparing the nucleic acid releasing compositions of the present invention (e.g., Olson, et al., Biophys. Acta, 557:9 (1979)). For example, prior art liposomal formulations used to encapsulate hemoglobin (e.g., U.S. Pat. No. 4,911,929) are to produce liposomal vesicles as described herein. Such liposomal formulation contains roughly equivalent quantities of cholesterol and phosphatidylcholine, with 5 to 10% negatively charged lipid, such as phosphatidic acid, dicetyl phosphate, or dimyristoyl phosphatidyl glycerol (DMPG). Hydration of the dried lipid film results in formation of multi-lamellar vesicles (MLV), which can be extruded at low-pressure (e.g., 50-90 psi) through filters of progressively smaller pore size to large unilamellar vesicles (LUVs). Once the liposomal vesicles are formed, any unencapsulated aqueous solution can be removed, if desired, by centrifugation or diafiltration and then recycled.
Lipid used for the formation of the liposome can be natural or synthetic and include phospholipids, glycolipids, and lipid related compounds. Exemplary lipids include, lecithin (phosphatidylcholine), phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, cardiolipin, and hydrogenated derivatives thereof, which can be used either alone or in combinations. The glycolipids include cerebroside, sulfolipid (e.g., sulfatide), and ganglioside. The structure of the liposomes may be as a multilamellar vesicle (MLV), a small unilamellar vesicle (SUV), or large unilamellar vesicle (LUV).
To stabilize the lipid, an antioxidant such as tocopherol (vitamin E) can be added to the solution. A suitable amount of an antioxidant is about 0.01 to 0.5% by weight based on the weight of the phospholipid. The liposome composition of the invention also can contain, as a stabilizer, a high molecular weight polymer such as albumin, dextran, vinyl polymers, non-ionic surface active agents, gelatin, and hydroxyethyl starch.
Lipid used for the formation of the liposome can be natural or synthetic and include phospholipids, glycolipids, and lipid related compounds. Exemplary lipids include, lecithin (phosphatidylcholine), phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, cardiolipin, and hydrogenated derivatives thereof, which can be used either alone or in combination. The glycolipids include cerebroside, sulfolipid (e.g., sulfatide), and ganglioside. The structure of the liposomes may be as a multilamellar vesicle (MLV), a small unilamellar vesicle (SUV), or large unilamellar vesicle (LUV).
To stabilize the lipid, an antioxidant such as tocopherol (vitamin E) can be added to the solution. A suitable amount of an antioxidant is about 0.01 to 0.5% by weight based on the weight of the phospholipid. The liposome composition of the invention also can contain as a stabilizer, a high molecular weight polymer such as albumin, dextran, vinyl polymers, non-ionic surface active agents, gelatin, and hydroxyethyl starch.
Liposomal vesicles that encapsulate stabilized cocktails as used herein can be prepared by a variety of known methods. For example, conventionally used hydration, reversed phase evaporation, removal of surfactant, solvent injection, freeze-thawing and dehydration-rehydration can be employed.
In the hydration method, the selected lipids are dissolved in an organic solvent (e.g., chloroform and ether), which is non-denaturing, and the solvent is evaporated from the resulting solution yield a thin homogeneous film. An aqueous solution containing the stabilized cocktail is added to the thin membrane, and the mixture is subjected to agitation and sonication to yield a liposome preparation encapsulating the aqueous solution. The aqueous solution contains a buffer at a pH between 4 and 11. The pH of the buffer is chosen such that when the lipids or liposomes are added to an assay medium, the final pH in a range suitable to preserve nucleic acids in solution.
In the reversed-phase evaporation method, the selected lipids are dissolved in an organic solvent (e.g., chloroform and ether), as discussed above, and are added to the aqueous solution containing the stabilized cocktail and subjected to agitation, sonication and high pressure homogenization to uniformly disperse the aqueous solution. The solvent is evaporated from this dispersion to yield a liposome preparation encapsulating the aqueous solution.
In the removal of surfactant approach, the selected lipids dissolved in organic solvent are mixed with a surfactant (e.g., cationic surfactant such as cholic acid or deoxycholic acid, and a non-ionic surfactant such as Triton X-100 and octyl-D-glucoside) and added to the aqueous solution containing the stabilized cocktail, which is followed by agitation, sonication and high pressure homogenization to uniformly disperse the aqueous solution. The surfactant is then removed by dialysis, gel filtration and ultrafiltration, which are applied singly or in combination.
In the solvent injection, approach, the selected lipids are dissolved in organic solvent and are added to the aqueous solution containing the stabilized cocktail, which has been set for a temperature about 10xc2x0 C. higher than the boiling point of the organic solvent. Then, the organic solvent is evaporated.
A composition of the invention comprising a stabilized nucleic acid amplification cocktail also can comprise reagents useful for releasing nucleic acid from a cell sample in a form suitable for directly detecting the nucleic acid as described in U.S. Provisional Patent Application entitled xe2x80x9cSample Processing to Release Nucleic Acids for Direct Detectionxe2x80x9d by Dattagupta et al., filed Jul. 30, 1999. The a reagent cocktail can include primers, enzymes, nucleoside triphosphates, deoxynucleoside triphosphates and other components as needed for amplification and appropriate reagents to release the nucleic acid. In this approach, a single addition of the stabilized cocktail with the lipid reagents can be added to a cell sample and release and amplification of a target nucleic acid can be achieved without further reagent addition. This can be accomplished because the added lipids are non-denaturing and non-inhibitory of nucleic acids or proteins used in nucleic acid release, amplification, labeling or detection.
Reagents useful for releasing nucleic acid without denaturation include an aqueous solution that comprises a water and/or other water miscible solvent and further includes a buffer to stabilize the pH between 4 and11, with the ultimate pH depending on the stability of the nucleic acid to be released.
The aqueous solution comprising one or more lipids or a liposomal formulation includes those lipids suitable for releasing cellular or otherwise inaccessible nucleic acid without denaturation. Liposomal formulations containing cationic lipids that have been used for delivery of oligonucleotides and other agents to target cells are useful for releasing nucleic acid from cells without denaturation as provided herein. PCT WO 96/40627 and U.S. Pat. Nos. 5,851,548, 5,759,519, 5,756,352, and 5,739,271 teach liposomal formulations containing cationic lipids.
The lipid containing vesicles or other structures used in the present compositions for releasing nucleic acid from cells include complex mixtures of different lipophilic substituents. Such complex mixtures allow for optimization of the physical properties of the liposomes, such as pH sensitivity, temperature sensitivity and size. For example, in certain embodiments, dioleoylphosphatidylethanolamine (xe2x80x9cDOPExe2x80x9d), and other pH sensitive amphiphilic compounds can be used to formulate liposomes which destabilize at acidic pH. This promotes fusion of the liposome with endosomal membranes when exposed to the degradative acidic pH and enzymatic contents of the endosome, resulting in release of the contents of the endosome into the cytoplasm. (Ropert, et al., Biochem. Biophys. Res. Comm. 183(2):879-895 (1992); Juliano, et al., Antisense Res. and Dev. 2:165-176 (1992)). Although not wishing to be bound by any particular theory, it is believed that pH controlled degradation of liposomes in the cytoplasm of the cell enhances release of nucleic acids.
Lipids for releasing nucleic acid from cells also can include sterols to enhance stability of liposomal vesicles both in vitro and in vivo. In particular, organic acid derivatives of sterols, such as cholesterol or vitamin D3, which have been reported to be easier to formulate than their non-derivatized water-insoluble equivalents (e.g., U.S. Pat. Nos. 4,721,612 and 4,891,208), are useful in preparing liposomal formulations as described herein.
Preferred lipids for use in the present compositions and methods are cationic lipids (i.e., derivatives of glycerolipids with a positively charged ammonium or sulfonium ion-containing headgroup), including those useful in liposomal formulations for the intracellular delivery of negatively charged biomolecules such as oligonucleotides. The usefulness of cationic lipids may be derived from the ability of their positively charged headgroups to interact with negatively charged cell surfaces, although this is not known for certain. The cationic lipid N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (xe2x80x9cDOTMAxe2x80x9d) as described by Felgner, et al., Proc. Natl. Acad. Sci. (USA) 84:7413-7417 (1987) (see also U.S. Pat. No. 4,897,355) is a cationic lipid with an ammonium group that can be used in lipid formulations in the compositions of the invention. In such formulations, DOTMA may bind to DNA through an ionic lipid-DNA complex that assists in releasing nucleic acid from a cell.
Other ammonium ion-containing cationic lipid formulations that can be used in the nucleic acid releasing compositions of the present invention include the DOTMA analog, 1,2-bis(oleoyloxy)-3(trimethylammonio)propane (xe2x80x9cDOTAPxe2x80x9d) (Stamatatos, et al., Biochemistry, 27:3917-3925 (1988)); the 5lipophilic derivative of spermine (Behr, et al., Proc. Natl. Acad. Sci. (USA), 86:6982-6986 (1989)); and cetyltrimethylammonium bromide (Pinnaduwage, et al., Biochem. Biophys. Acta, 985:33-37 (1989); see Leventis, et al., Biochem. Biophys. Acta, 1023:124-132 (1990); Zhou, et al., Biochem. Biophys. Acta, 1065:8-14 (1991); Farhood, et al., Biochem. Biophys. Acta, 1111:239-246 (1992); and Gao, et al., Biochem. Biophys. Res. Comm., 179:280-285 (1991)). Cationic lipids are commercially available including DOTMA (Gibco BRL, Bethesda, Md.), DOTAP (Boehringer Mannheim, Germany), and 1,2-diacyl-3-trimethylammonium propane (xe2x80x9cTAPxe2x80x9d) (Avanti Polar Lipids, Alabaster, Ala.).
Cationic lipids containing sulfonium ions (i.e., thiocationic lipids) also can be used in combination with a stabilized cocktail to release nucleic acid. Sulfonium ions have entirely different physical properties than ammonium ions, which provides sulfonium cationic lipids with some unique properties. Ammonium ion-containing compounds are classified as hard bases, because the nitrogen atom possesses high electronegativity, is difficult to polarize and oxidize, and the valence electrons are held tightly by the nucleus. This characteristic may account for some of the toxicity associated with ammonium ion-containing lipid formulations. In contrast, sulfonium ion-containing compounds are classified as soft bases, because the sulfur atom possesses low electronegativity, is easy to polarize and oxidize, and the valence electrons are held more loosely by the nucleus. This decreased charge density exhibited by sulfonium ion-containing (i.e. xe2x80x9cthiocationicxe2x80x9d) lipids may effectuate an enhanced interaction with negatively charged cellular membranes, as well as a decreased toxicity, leading to compositions with increased ability to release cell nucleic acid in a non-denatured form.
Cationic lipids with relatively small polar headgroups as described by Felgner, et al., J. Biol. Chem., 269(4):2550-2561 (1994), can be particularly useful in the present compositions for releasing nucleic acids. However, the sulfonium ion type cationic lipid, which has a relatively larger headgroup, also can be useful because of the physiochemical properties associated with the sulfonium ion. A lipid headgroup that consists of a sulfur atom surrounded by adjoining saturated carbon atoms exhibits a diffusion of charge to the neighboring carbon atoms that can facilitate interaction of the lipid with cellular membranes, as well as decrease the toxicity of the lipid (U.S. Pat. No. 5,759,519).
The liposome preparations used in combination with the stabilized cocktail of reagents for amplification also can include a positively charged surface by including in the formulation, saturated or unsaturated aliphatic amines including, e.g., stearylamine and oleylamine, sphingosine, phosphatidylethanolamine, N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammoniumchloride, cholesterylhemisuccinate, 3xcex2-(N-(Nxe2x80x2,Nxe2x80x2-dimethylaminoethane)carbamoyl)cholesterol and cholesteryl(4xe2x80x2-trimethylammonio)butanoate, with preference given to stearylamine and sphingosine as described in U.S. Pat. No. 5,759,519.
The stabilized cocktail including reagents for releasing nucleic acid also can include, for example, substances other than lipids that enhance release of nucleic acid depending on the nature of the sample and the environment in which the nucleic acid is contained (e.g., the type of cell). Such nucleic acid releasing substances include, for example, an enzyme(s) to degrade cell structure, a non-ionic membrane fluidizing compound(s), and/or a metal chelator(s).
Enzymes suitable for use with lipid containing aqueous solution are available from natural sources or produced by recombinant DNA methods. Such enzymes include, for example, lysozyme, lipases, and proteinases such as proteinase K, pronase, trypsin and chymotrypsin. Lysozymes from bovine, chicken, human and lipases from wheat germ, human, yeast and other sources also are suitable enzymes to degrade cell structure. These enzymes preferably are nuclease free to support stability of released nucleic acids in solution. The aqueous solution containing lipids and enzymes for releasing nucleic acid can be encapsulated into a liposome, if desired.
The enzymes are used at a molar ratio of lipid to enzyme of between 10,000: 1 and 1:10,000. The optimal ratio of enzyme to lipid can be readily determined by one skilled in the art. This can be accomplished by mixing target cells with various lipid:enzyme ratios, and determining the effectiveness of releasing nucleic acid in a probe hybridization assay.
Non-ionic membrane fluidizing compounds, which have been described in Suciu et al., Mol. Microbiol., 21:181-95 (1996), Nabekura, et al., Pharm. Res., 13(7):1069-72 (1996), and Lindow, et al., Cryobiol., 32(3):247-258 (1995), and include aromatic alcohols such as all phenyl, napthyl, and higher alcohols, also can be used to release nucleic acid from cells without denaturation of enzymes or proteins. The hydrocarbon side chains of aromatic alcohols can be from C1to C50 and longer, preferably between C1 and C20. The xe2x80x94OH residue can be at the Cn terminus carbon for a primary alcohol or any place as in a secondary or tertiary alcohol. The Cxe2x80x94C bonds in Cn chain in addition to single bond can have unsaturated linkages in the form of double or triple bonds. The carbon chain also can have secondary and tertiary C-linkages. Phenethyl alcohol, sec-phenethyl alcohol, benzyl alcohol are examples of non-ionic membrane fluidizing compounds.
Non-ionic membrane fluidizing compounds can be included in the stabilized cocktail of reagents so as to enhance release of nucleic acids from cells without creating an enzyme or protein inhibitory environment. Such compounds can be present in the aqueous solution at a concentration between 0.001% and 10.0% . The final concentration of non-ionic membrane fluidizing compound in a sample for releasing nucleic acid is preferably between about 0.001 and 10% (v/v), more preferably between 0.01% and 5%, most preferably between 0.1% and 2%. The ultimate concentration of the non-ionic membrane fluidizing compound depends on the nature of the fluidizing compound and the other components of the nucleic acid releasing composition. One skilled in the art can readily determine the proper concentration of membrane fluidizing compound for effective release of nucleic acid from a particular sample by determining binding of a specific probe to nucleic acid released by a particular formulation.
The aqueous solution of the nucleic acid releasing composition also can include metal chelators such as ethylenediaminetetraacetic acid (EDTA) and ethyleneguaninetetraacetic acid (EGTA). In addition, the aqueous solution can be heated to enhance release of the nucleic acid essentially as described in U.S. Pat. No. 5,837,452.
The present invention also provides methods for preparing a stabilized cocktail of reagents which avoids undesirable reactions during storage and for using such compositions for amplifying a nucleic acid. The stabilized cocktail of reagents, as discussed above, is useful for amplification in virtually any amplification format, including, for example, the polymerase chain reaction, ligase chain reaction, transcription based amplification reaction, nucleic acid sequence based amplification reaction and strand displacement amplification reaction.
Amplification methods suitable for use with the present methods include, for example, polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription mediated amplification (TMA) reaction, nucleic acid sequence based amplification (NASBA) reaction, and strand displacement amplification (SDA) reaction. These methods of amplification are well known in the art.
PCR can be performed as according to Whelan, et al., J. Clin. Microbiol., 33(3):556-561 (1995). Briefly, a PCR reaction mixture includes two specific primers, dNTP, 0.25 Units (U) of Taq polymerase, and 1xc3x97PCR Buffer. For every 25 xcexcl PCR reaction, a 2 xcexcl sample (e.g., isolated DNA from target organism) is added and amplified on a thermal cycler. The amplification cycle includes an initial denaturation, and up to 50 cycles of annealing, strand elongation and strand separation (denaturation).
LCR can be performed as according to Moore et al., J. Clin. Microbiol., 36(4):1028-1031 (1998). Briefly, an LCR reaction mixture contains two pair of probes, dNTP, DNA ligase and DNA polymerase representing about 90 xcexcl, to which is added 100 xcexcl of isolated nucleic acid from the target organism. Amplification is performed in a thermal cycler (e.g., LCx(copyright) thermal cycler, Abbott Labs, North Chicago, Ill.).
SDA can be performed as according to Walker et al., Nucleic Acids Res., 20(7):1691-1696 (1992). Briefly, an SDA reaction mixture contains four SDA primers, dGTP, dCTP, TTP, dATPS, 150 U of Hinc II, and 5 U of exonuclease deficient E. coli DNA polymerase I. The sample mixture is heated 95xc2x0 C. for 4 minutes (min) to denature target DNA prior to addition of the enzymes. After addition of the two enzymes, amplification is carried out for 120 min at 37xc2x0 C. in a total volume of 50 xcexcl. The reaction is terminated by heating for 2 minutes at 95xc2x0 C.
NASBA can be performed as according to Heim et al., Nucleic Acids Res., 26(9):2250-2251 (1998). Briefly, an NASBA reaction mixture contains two specific primers, dNTP, NTP, 6.4 U of AMV reverse transcriptase, 0.08 U of Escherichia coli Rnase H, and 32 U of T7 RNA polymerase. The amplification is carried out for 120 min at 41xc2x0 C. in a total volume of 20 xcexcl.
TMA can be performed as according to Wylie et al., J. Clin. Microbiol., 36(12):3488-3491 (1998). In TMA, nucleic acid targets are captured with magnetic beads containing specific capture primers. The beads with captured targets are washed and pelleted before adding amplification reagents, which contain amplification primers, dNTP, NTP, 2500 U of reverse transcriptase and 2500 U of T7 RNA polymerase. A 100 xcexcl TMA reaction mixture is placed in a tube, 200 xcexcl oil reagent is added and amplification is accomplished by incubation at 42xc2x0 C. in a waterbath for one hour (hr).
A variety of amplification enzymes are well known in the art and include, for example, DNA polymerase, RNA polymerase, reverse transcriptase, Q-beta replicase, thermostable DNA and RNA polymerases. Because these and other amplification reactions are catalyzed by enzymes, it is important for a single step assay that the nucleic acid releasing reagents and the detection reagents are not potential inhibitors of amplification enzymes if the ultimate detection is to be amplification based.
Also included in the composition for amplification are appropriate nucleoside triphosphates, amplification buffer and certain ions. The concentrations of nucleic acid primers and enzymes can be selected for specific use. For example, for polymerase chain reaction, the concentration of the nucleic acid primer is between 1 picomole and 1 millimole when added to the sample. The enzyme concentration can vary between about 0.01 U and 100,000 U. One skilled in the art can determine the optimal concentration of enzyme and other reagents by routine experimentation.
Detection of the nucleotide sequences also can be performed directly without amplification by hybridizing the sample nucleic acid to the nucleic acid probe present in the composition. In this case, the nucleic acid is contacted and incubated with the labeling reagents (provided in the nucleic acid release composition or separately) and the mixture is irradiated at a particular wavelength for the covalent interaction between the photochemically reactive DNA binding ligand and the test sample to take place. After labeling, the material is hybridized under specified hybridization conditions with a probe specific for the target nucleic acid.
Hybridization of the labeled sample nucleic acid or the labeled nucleic acid probe can be detected in any conventional hybridization assay format and, in general, in any format suitable for detecting the hybridized product or aggregate comprising the labeled nucleic acid. If the sample nucleic acid has been labeled, it can be used for hybridization in solution and solid-phase formats, including, in the latter case, formats involving immobilization of either sample or nucleic acid probe. For example, preimmobilized nucleic acid probe can be hybridized with labeled sample nucleic acid. The presence of label associated with the solid phase indicates hybridization between the probe and the sample nucleic acid and, thus, detection of the target nucleotide sequence. Alternatively, unlabeled sample nucleic acid can be preimmobilized and a labeled probe evaluated for hybridization thereto.
Preferable concentration for the probe is between about 0.01 picomole and 10 millimoles, more preferably between about 1 picomole and 1 millimole, and most preferably between about 10 picomole and 10 micromoles. Methods of detecting hybrids on solid phases are well known in the art and have been extensively described (e.g., U.S. Pat. Nos. 5,232,831, 4,950,613, 486,539 and 4,563,419).
The nucleic acid probe comprises at least one hybridizable, e.g., single-stranded, base sequence substantially complementary to or homologous with the nucleotide sequence to be detected. However, such base sequence need not be a single continuous polynucleotide segment, but can comprise two or more individual segments interrupted by non-homologous sequences. These non-homologous sequences can be linear or they can be self-complementary and form hairpin loops. In addition, the homologous region of the probe can be flanked at the 3xe2x80x2- and 5xe2x80x2 termini by non-homologous sequences, such as those comprising the DNA or RNA or a vector into which the homologous sequence had been inserted for propagation. In either instance, the probe as presented as an analytical reagent will exhibit detectable hybridization at one or more points with sample nucleic acids of interest. Linear or circular hybridizable, e.g., single-stranded polynucleotides can be used as the probe element, with major or minor portions being duplexed with a complementary polynucleotide strand or strands, provided that the critical homologous segment or segments are in single-stranded form and available for hybridization with sample DNA or RNA. Useful probes include linear or circular probes wherein the homologous probe sequence essentially is a single-stranded form (Hu et al., Gene, 17:271 (1982)).
The nucleic acid probe can be used in any conventional hybridization technique. As improvements are made and conceptually new formats are developed, such can be readily applied to the present probes. Conventional hybridization formats that are particularly useful include those wherein the sample nucleic acids or the polynucleotide probe are immobilized on a solid support (solid-phase hybridization) and those wherein the polynucleotide species are all in solution (solution hybridization).
In solid-phase hybridization formats, one of the polynucleotide species participating in hybridization is fixed in an appropriate manner in its single-stranded form to a solid support. Useful solid supports are well known in the art and include those, for example, which bind nucleic acids either covalently or non-covalently. Non-covalent binding supports, which are generally understood to involve hydrophobic bonding include naturally occurring and synthetic polymeric materials, such as nitrocellulose, derivatized nylon and fluorinated polyhydrocarbons, in a variety of forms such as filters, beads or solid sheets. Covalent binding supports (in the form of filters, beads or solid sheets, just to mention a few) are also useful and comprise materials having chemically reactive groups or groups such as dichlorotriazine, diazobenzyloxymethyl, and the like, which can be activated for binding to polynucleotides.
It is well known that non-covalent immobilization of an oligonucleotide to a solid support such as nitrocellulose paper is generally ineffective for detecting hybridization. Thus, covalent immobilization is preferred and can be achieved by phosphorylation of an oligonucleotide by a polynucleotide kinase or by ligation of the 5xe2x80x2-phosphorylated oligonucleotide to produce multioligonucleotide molecules capable of immobilization. The conditions for kinase and ligation reaction have been described previously (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1.53 and 5.33, (1989). Thus oligonucleotide probes specific for genetic defects related to hemoglobinopathies, such as sickle cell anemia and alpha-thalassemias can be immobilized on nitrocellulose paper and contacted with patient sample nucleic acid labeled by the above described method. The photochemical labeling can be done in a single step without the need to obtain purified nucleic acid samples and without affecting the specific hybridizability of the labeled sample.
A typical solid-phase hybridization technique begins with immobilization of sample nucleic acids onto the support in single-stranded form. This initial step essentially prevents reannealing of complementary strands from the sample and can be used for concentrating sample material on the support for enhanced delectability. The nucleic acid probe is then contacted with the support and hybridization detected by measurement of the label as described herein. The solid support provides a convenient means for separating labeled probe, which has hybridized to the sequence to be detected, from probe that has not hybridized.
Another method of interest is the sandwich hybridization technique wherein one of two mutually exclusive fragments of the homologous sequence of the probe is immobilized and the other is labeled. The presence of the polynucleotide sequence of interest results in dual hybridization to the immobilized and labeled probe segments (Rankim, et al., 21:77-85 (1983)).
In one embodiment, the immobile phase of the hybridization system can be a series or matrix of spots of known kinds and/or dilutions of denatured DNA. This can be prepared by pipetting appropriate small volumes of native DNA onto a dry nitrocellulose or nylon sheet, floating the sheet on a sodium hydroxide solution to denature the DNA, rinsing the sheet in a neutralizing solution, then baking the sheet to fix the DNA. Before DNA:DNA hybridization, the sheet is usually treated with a solution that inhibits non-specific binding of added DNA during hybridization.
In solid phase detection systems, unhybridized labeled test sample can be removed by washing following hybridization. After washing, the hybrid is detected through the label carried by the test sample, which is specifically hybridized with a specific probe.
In some embodiments, the composition of stabilized cocktail can include reagents to label the released nucleic acid for later detection of formed hybrids essentially as described in U.S. Pat. Nos. 4,950,744 and 5,026,840. Such reagents for labeling nucleic acid comprise a binding ligand comprising a chemical moiety that binds to a nucleic acid and that, when activated by light (i.e. photochemistry), forms at least one covalent bond therewith, a label comprising a detectable moiety and optionally, a binding enhancer comprising a chemical moiety that has a specific affinity for nucleic acids (U.S. patent application Ser. No. 09/265,127, now U.S. Pat. No. 6,187,566 B1, issued Feb. 13, 2001). Covalent or non-covalent complexes of a binding ligand, a binding enhancer and a label is referred to herein as a xe2x80x9cLAC.xe2x80x9d
The nucleic acid binding enhancer (xe2x80x9cbinding enhancerxe2x80x9d), serves to enhance the affinity of the LAC for nucleic acids above that exhibited with the binding ligand alone. Accordingly, binding enhancers tend to have a specific affinity for nucleic acids when compared to non-nucleic acid sample/reaction constituents. The binding enhancer can be the same as or different from the binding ligand. In other words, the binding ligand and the binding enhancer can each be an intercalator, wherein one of the two is a monoadduct-forming species, and the other is present to enhance binding by this monoadduct-forming species. Examples of such xe2x80x9cdual rolexe2x80x9d binding ligands are described in Chaires, et al., J. Med. Chem., 40:261-266 (1977). Therein, it has been described that binding of a bis-intercalating anthracycline antibiotic reached as high as 1011 at 20xc2x0 C. It was also shown that the affinity of a similar monointercalator is not above 107 (Chaires, et al., Biochem., 35:2047-2053 (1996)).
The binding enhancer also can be a non-intercalating compound. There are many non-intercalating nucleic acid binding molecules known in the art. A bis-benzimidazole derivative commonly known as Hoechst 33258 has shown affinity as high as 3.2xc3x97108 Mxe2x88x921 (Haq, et al., J. Mol. Biol., 271:244-257 (1997)). Other non-intercalating binding enhancers are oligo pyrroles, phenyl indole derivatives and the like. These molecules do not bind nucleic acids solely on the basis of positive charge. Other suitable binding enhancers bind nucleic acids on the basis of hydrogen bond formation, hydrophobic interaction in the major or minor groove of the nucleic acid double helix and other non-ionic interactions that give rise to high affinity reactions with nucleic acids.
Not every compound capable of forming an electrostatic bond with a negatively charged nucleic acid can serve as a binding enhancer. For example, polycations such as polyamines are generally not suitable for use in the present invention because of their inability to specifically bind to nucleic acids in crude samples and in the presence of amplification reaction components. Such positively charged compounds can, for example, non-specifically bind to all anionic macromolecules present in the sample, and not just to nucleic acids. In addition, the binding enhancer should be capable of specifically binding to nucleic acids in the presence of 10 to 20 mM magnesium, which is typically required for most amplification reactions. At this concentration, compounds that bind to nucleic acids solely on the basis of electrostatic interactions do not form stable complexes with nucleic acids and thus require a greater concentration of LAC for efficient labeling.
The binding ligand for labeling nucleic acid is either directly or indirectly linked to a label essentially as described in U.S. Pat. Nos. 4,950,744 and 5,026,840. Certain compounds can serve the dual role of a binding enhancer and a linker. For example, linkers can be constructed from positively charged compounds, such that they enhance binding with negatively charged nucleic acids. However, in order for the linker to also serve as a binding enhancer, it is necessary for it to have a specific affinity for nucleic acids, and not just a structure specific electrostatic affinity for negatively charged compounds. The polyalkylamine linkers described in U.S. Pat. No. 5,026,840 are especially useful as binding enhancers, although they can be suitable for use as linkers.
In a preferred embodiment, a bifunctional linker is used that is capable of reacting with both the nucleic acid binding moiety and the label to form a chemical bridge therebetween. However, in an alternate embodiment, a multifunctional linker can be employed, to which the binding ligand, the binding enhancer and the label are attached as a xe2x80x9cbranchedxe2x80x9d complex. Such complex formats and chemical reactions for forming these types of complexes are well known in the art.
The present invention also provides methods and kits for using the disclosed compositions in assays for detecting the presence of a nucleotide sequence in nucleic acid of a sample containing cells. Such kits may also include other materials that would make the invention a part of other procedures including adaptation to multi-well technologies. The items comprising the kit may be supplied in separate vials or may be mixed together, where appropriate.
The compositions, methods and kits of the present invention can be used in assays for diagnosis of infectious diseases, cancer, human genetic disorders, and others like histocompatibility (e.g., HLA) typing, forensic and paternity testing. For example, a clinical sample can be contacted with the above described compositions which include a stabilized cocktail of amplification reagents and diagnosis of infectious disease determined. The stabilized cocktail also can include reagents for releasing nucleic acid from cells and appropriate labeling reagents (e.g., LACs) such that the clinical sample can be diagnosed without any further reagent addition. Thus, a urine sample, for instance, that is suspected of bacterial infections can be labeled without centrifugation, filtration or dialysis and the cells in the samples are lysed without any separation step.
Test samples include body fluids, e.g., urine, blood, semen, cerebrospinal fluid, pus, amniotic fluid, tears, or semisolid or fluid discharge, e.g., sputum, saliva, lung aspirate, vaginal or urethral discharge, stool or solid tissue samples, such as a biopsy or chorionic villi specimens. Test samples also include samples collected with swabs from the skin, genitalia, or throat. The compositions of the invention can be added directly to the sample or to cells isolated from the sample.
The assay method can detect the nucleic acid from essentially any species of organism, including, for example, Acintobacter, Actinomyces, Aerococcus, Aeromonas, Alclaigenes, Bacillus, Bacteriodes, Bordetella, Branhamella, Bevibacterium, Campylobacter, Candida, Capnocytophagia, Chlamydia, Chromobacterium, Clostridium, Corynebacterium, Cryptococcus, Deinococcus, Enterococcus, Erysielothrix, Escherichia, Flavobacterium, Gemella, Gonorrhea, Haemophilus, Klebsiella, Lactobacillus, Lactococcus, Legionella, Leuconostoc, Listeria, Micrococcus, Mycobacterium, Neisseria, Nocardia, Oerskovia, Paracoccus, Pediococcus, Peptostreptococcus, Propionibacterium, Proteus, Psuedomonas, Rahnella, Rhodococcus, Rhodospirillium, Staphlococcus, Streptomyces, Streptococcus, Vibrio, and Yersinia. Also included are viruses such as the hepatitis viruses and human immunodeficiency viruses (HIV).
The present methods also can be used to detect nucleic acid from eukaroytes (protists) in samples from higher organisms, such as animals or humans. Eukaroytes include algae, protozoa, fungi and slime molds. The term xe2x80x9calgaexe2x80x9d refers in general to chlorophyll-containing protists, descriptions of which are found in Smith, Cryptogamic Botany, 2nd ed. Vol. 1, Algae and Fungi, McGraw-Hill, (1955). Eukaryotic sequences according to the present invention includes all disease sequences. Accordingly, the detection of genetic diseases, for example, also are embraced by the present invention.
Methods of detecting a nucleotide sequence involve contacting the sample with above described aqueous compositions of a stabilized cocktail and reagents for releasing nucleic acid. The mixture is incubated for an appropriate period of time and under conditions suitable for releasing the nucleic acid from the cells. If the sample already contains released or isolated nucleic acid, only the stabilized cocktail of reagents for amplification need be added.